This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Beclin 1 is a mammalian autophagy protein which was shown to play an important role in development, tumor suppression, neurodegeneration and cell death. Beclin 1 forms a complex with Vps34/phosphatidylinositol (PtdIns) 3-kinase (class III PI-3K), which mediates multiple vesicle trafficking pathways including endocytosis and autophagy. However, the precise role of Beclin 1-Vps34/PtdIns3K complex in the regulation of autophagy remains to be elucidated. Through a study that combines mouse genetics and biochemistry, we uncover several protein components that associate specifically with Beclin 1 complexes in mouse tissues. The biochemical analysis reveals a large in vivo Beclin 1 complex containing the known proteins Vps34/PtdIns3K, p150/Vps15 and UVRAG, as well as novel proteins BISC and BIRC. Characterization of the novel proteins suggests that BISC and BIRC modulate Vps34/PtdIns3K activity in opposite manner. This opposing effect of BISC and BIRC on the lipid kinase activity is correlated with their distinct regulation of autophagy: BISC positively regulates autophagy, whereas BIRC is involved in negative control of autophagy. Moreover, we find that Beclin 1 and BISC synergistically promote formation of double membrane vacuoles which are associated with Atg5/Atg12, while forced expression of BIRC results in aberrant late endosomal/lysosomal structures (independent of Beclin 1) and impaired autophagosome maturation. Our study suggests that, by forming distinct protein complexes, Beclin 1 and its binding proteins orchestrate the precise function of Vps34/PtdIns3K in regulating autophagy at multiple steps. A manuscript describing this work has been accepted for publication in Nature Cell Biology